Carotenoids represent one of the most widely distributed and structurally diverse classes of natural pigments, producing pigment colors of light yellow to orange to deep red. Eye-catching examples of carotenogenic tissues include carrots, tomatoes, red peppers, and the petals of daffodils and marigolds. Carotenoids are synthesized by all photosynthetic organisms, as well as some bacteria and fungi. These pigments have important functions in photosynthesis, nutrition, and protection against photooxidative damage. For example, animals do not have the ability to synthesize carotenoids but must instead obtain these nutritionally important compounds through their dietary sources.
Industrially, only a few carotenoids are used for food colors, animal feeds, pharmaceuticals, and cosmetics, despite the existence of more than 600 different carotenoids identified in nature. This is largely due to difficulties in production. Presently, most of the carotenoids used for industrial purposes are produced by chemical synthesis; however, these compounds are very difficult to make chemically (Nelis and Leenheer, Appl. Bacteriol. 70:181–191 (1991)). Natural carotenoids can either be obtained by extraction of plant material or by microbial synthesis, but only a few plants are widely used for commercial carotenoid production and the productivity of carotenoid synthesis in these plants is relatively low. As a result, carotenoids produced from these plants are very expensive.
Structurally, the most common carotenoids are 40-carbon (C40) terpenoids; however, carotenoids with only 30 carbon atoms (C30; diapocarotenoids) are detected in some species. Biosynthesis of each of these types of carotenoids are derived from the isoprene biosynthetic pathway and its five-carbon universal isoprene building block, isopentenyl pyrophosphate (IPP). This biosynthetic pathway can be divided into two portions: 1) the upper isoprenoid pathway, which leads to the formation of farnesyl pyrophosphate (FPP); and 2) the lower carotenoid biosynthetic pathway, comprising various crt genes which convert FPP into long C30 and C40 carotenogenic compounds characterized by a long central chain of conjugated double bonds. It is the degree of the carbon backbone's unsaturation, conjugation and isomerization that determines the specific carotenoids' unique absorption characteristics and colors.
Various other crt genes are known, which enable the intramolecular conversion of linear C30 and C40 compounds to produce numerous other functionalized carotenoid compounds by: (i) hydrogenation, (ii) dehydrogenation, (iii) cyclization, (iv) oxidation, (v) esterification/glycosylation, or any combination of these processes.
Finally, Halobacterium cutirubrum is known to produce both C40 and C50 carotenoids, as well as a C30 diapophytoene (Kushwaha, S. C., et al., Biochim. Biophys. Acta. 260: 492–506 (1972)). Other desaturases are likely to soon be developed in laboratories through mutational techniques, to create completely novel carotenoid pathways, up to C80. These large carotenoids can potentially undergo many desaturation steps and generate large chromophores for purple or blue-purple pigmentation. Such novel carotenoids have potential applications as strong antioxidants or colorants for foods and cosmetics.
The genetics of C40 carotenoid pigment biosynthesis has been extremely well studied in the Gram-negative pigmented bacteria of the genera Pantoea, formerly known as Erwinia. In both E. herbicola EHO-10 (ATCC 39368) and E. uredovora 20D3 (ATCC 19321), the crt genes are clustered in two genetic units, crt Z and crt EXYIB (U.S. Pat. No. 5,656,472; U.S. Pat. No. 5,545,816; U.S. Pat. No. 5,530,189; U.S. Pat. No. 5,530,188; U.S. Pat. No. 5,429,939). These genes have subsequently been sequenced and identified in a suite of other species of bacterial, fungal, plant and animal origin. Several reviews discuss the genetics of carotenoid pigment biosynthesis, such as those of Armstrong (J. Bact. 176: 4795–4802 (1994); Annu. Rev. Microbiol. 51:629–659 (1997)).
The abundant knowledge concerning the genetics of C40 biosynthesis has permitted production of a number of natural C40 carotenoids from genetically engineered microbial sources. Examples include:                1.) Lycopene (Farmer W. R. and J. C. Liao. Biotechnol. Prog. 17: 57–61(2001); Wang C. et al., Biotechnol Prog. 16: 922–926 (2000); Misawa, N. and H. Shimada. J. Biotechnol. 59: 169–181 (1998); Shimada, H. et al. Appl. Environ. Microbiol. 64:2676–2680 (1998));        2.) β-carotene (Albrecht, M. et al., Biotechnol. Lett. 21: 791–795 (1999); Miura, Y. et al., Appl. Environ. Microbiol. 64:1226–1229 (1998); U.S. Pat. No. 5,691,190);        3.) Zeaxanthin (Albrecht, M. et al., Biotechnol. Lett. 21: 791–795 (1999); Miura, Y. et al., Appl. Environ. Microbiol. 64:1226–1229 (1998)); and        4.) Astaxanthin (U.S. Pat. No. 5,466,599; U.S. Pat. No. 6,015,684; U.S. Pat. No. 5,182,208; U.S. Pat. No. 5,972,642).Genes encoding various elements of the lower C40 carotenoid biosynthetic pathway have been cloned and expressed in various microbes (U.S. Pat. No. 5,656,472; U.S. Pat. No. 5,545,816; U.S. Pat. No. 5,530,189; U.S. Pat. No. 5,530,188; U.S. Pat. No. 5,429,939; U.S. Pat. No. 6,124,113).        
Despite abundant knowledge and understanding of the C40 carotenoid pathway, C30 pigment biosynthesis is both less well understood and less prevalent in nature. Early studies by Kleinig, H. et al. (Z. Naturforsch 34c: 181–185 (1979); Z. Naturforsch 37c: 758–760 (1982)) examined the structure of C30 carotenoic acid glucosyl esters produced in Pseudomonas rhodos (subsequently renamed Methylobacterium rhodinum) and the biosynthesis of those compounds, according to analysis of mutants. To date, presence of diapocarotenoids has been discovered in Streptococcus faecium (Taylor, R. F. and B. H. Davies. J. Biochem. 139:751–760 (1974)), M. rhodinum (Kleinig, H. et al., supra; Taylor, R. F. Microbiol. Rev. 48:181–198 (1984)), genera of the photosynthetic heliobacteria (Takaichi, S. et al. Arch. Microbiol. 168: 277–281 (1997)), and Staphylococcus aureus (Marshall, J. H., and G. J. Wilmoth. J. Bacteriol., 147:900–913 (1981)). All appear to have a diapophytoene precursor, from which all subsequent C30 compounds are produced.
The relevant genes responsible for C30 carotenoid pigment biosynthesis are known to include crtM and crtN in Staphylococcus aureus. The diapophytoene desaturase CrtN can function to some extent in the C40 pathway, and the phytoene desaturase CrtI of the C40 carotenoids can also work in the C30 pathway (Raisig and Sandmann, Biochim. Biophys. Acta, 1533:164–170 (2001)). Investigators J. H. Marshall and G. J. Wilmonth (J. Bacteriol. 147:914–919 (1981)) suggest that mixed-function oxidases are responsible for the introduction of oxygen functions to produce the aldehyde and carboxylic acid of 4,4-diaponeurosporene. However, genes responsible for the addition of functionality to the terminal methyl group of the linear carotenoid molecule have not yet been identified, despite characterization of the resulting carotenoids. Genes in other organisms that produce C30 carotenoids have not been identified.
It is clear that scientific understanding has yet to reveal all of nature's untapped diversity, in order to industrially duplicate the wide spectrum of carotenoids that can be readily produced by nature. In light of these needs, the problem to be solved is to isolate and functionally characterize the nucleic acid sequences of those genes involved in C30 carotenoid biosynthesis in Methylomonas for their use in carotenoid production. Although some of the genes involved in the C30 carotenoid biosynthetic pathway are known in some organisms, understanding of the pathway is not complete. Genes involved in the C30 carotenoid biosynthesis of methylotrophic bacteria are not described in the existing literature, despite the existence of many pigmented methylotrophic and methanotrophic bacteria. Further, it is necessary to gain functional knowledge of reactions catalyzed by carotenoid enzymes and identify novel carotenoid genes.
Applicants have solved the stated problem by isolating and functionally characterizing three unique open reading frames encoding the enzymes crtN, ald, and crtN2 from a Methylomonas sp. strain 16a. These genes will aid in synthesis of carotenoids beyond what is “known” in nature, to enable industrial synthesis and high levels of production of uniquely functionalized C30–C80 carotenoid compounds.